EAGER:A Novel Lab-on-a-Chip Concept for Characterization of Nanovesicles based on their Dielectric Properties

EAGER：基于介电特性表征纳米囊泡的新型芯片实验室概念

• 批准号: 2020112
• 负责人: Leyla Esfandiari
• 金额: $ 9.95万
• 依托单位: University of Cincinnati Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2020112&HistoricalAwards=false
• 关键词: EAGER; Novel; Lab; Chip; Concept

The objective of this project is to develop a rapid and accurate lab-on-a-chip device for characterization of nano-size vesicles based on their unique and intrinsic biophysical properties. The immediate focus of this project is to characterize small cell-secreted vesicles (exosomes) which are classified as circulating biomarkers associated with many types of disease including cancer, diabetes, cardiovascular, infectious, and neurodegenerative diseases. This research also has the potential to be expanded beyond exosomes to detect and study other small membrane bound vesicles including viruses. Thus, this high throughput detection tool can be utilized in a wide range of medical diagnosis and biomedical research which could potentially reduce the cost of healthcare by providing frequent, affordable, and early testing to patients. Furthermore this multidisciplinary project crosses the traditional boundaries between engineering, physics, and biomedical sciences which will provide students with a unique educational experience during their academic training. The undergraduate students will be trained and exposed to the field of micro- nanotechnology and biomedical sciences by closely working with the graduate students and being mentored by the principal investigator. The proposed project will also educate public about the importance of the field of micro-nanotechnologies for medicine through YouTube channel and “ThinkTV” program which will be broadcasting in Southwest Ohio. Exosomes are extracellular vesicles with diameters of ~30-120 nm, released from many cell types into the extracellular space. They are composed of a lipid bilayer membrane containing various receptors and tetraspanin proteins. They also encapsulate nucleic acids, proteins, and lipids in their lumen. Exosomes are promising biomarkers for several reasons: 1) they are highly abundant in all bodily fluids and therefore easily accessible; 2) their composition reflects their cellular origins and can therefore serve as indicators of pathology; and 3) they are stable. Also, it has been shown that exosomes secreted from different cellular origins, in particular pathogenic exosomes, undergo compositional changes and could have additional membrane receptors and/or elevated or suppressed levels of nucleic acids which can be associated with their total electric charges and dipoles. However, use of exosomes as biomarkers has been hampered by the lack of workable technologies to reliably isolate and rigorously characterize their unique properties in a timely manner. Although, some of the biophysical properties of exosomes such as size, density and morphology have been characterized before, their dielectric property which is associated with their unique compositional charges has not yet been investigated. The proposed label-free lab-on-a-chip device will utilize controllable electrokinetic forces across an array of borosilicate micropipettes to rapidly entrap exosomes at the tip of the pipettes and characterize the vesicles based on their unique dielectric properties by measuring their impedance. The impedance measurement will be conducted across an array of microelectrodes embedded in close proximity to the pipettes’ tips as an alternative current (AC) is applied at a wide range of frequency spectrum (500 KHz to 50 MHz). The AC field at a wide frequency range will polarize the bound and unbound charges associate with exosomes’ structure. The difference in impedance measurements will be linked to exosomes’ unique dielectric properties which includes their membrane capacitance and cytosolic conductance. Additionally, the impedance of exosomes secreted from different cellular origins and size distribution will be measured and their uniqueness in dielectric properties will be investigated. This rapid and label-free electrokinetic device can be further evolved as a diagnostic tool for initial non-invasive detection of pathogenic exosomes while keeping their compositions intact for further downstream analysisThis award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

本项目的目标是开发一种快速准确的芯片实验室设备，用于基于其独特的内在生物物理特性表征纳米尺寸的囊泡。该项目的直接重点是表征小细胞分泌的囊泡（外泌体），这些囊泡被归类为与许多类型的疾病相关的循环生物标志物，包括癌症，糖尿病，心血管，传染病和神经退行性疾病。这项研究也有可能扩展到外泌体之外，以检测和研究其他小的膜结合囊泡，包括病毒。因此，这种高通量检测工具可以用于广泛的医学诊断和生物医学研究，这可以通过为患者提供频繁的、负担得起的和早期的测试来潜在地降低医疗保健的成本。此外，这个多学科项目跨越了工程，物理和生物医学科学之间的传统界限，这将为学生在学术培训期间提供独特的教育体验。本科生将通过与研究生密切合作并由首席研究员指导来接受培训并接触微纳米技术和生物医学科学领域。拟议的项目还将通过YouTube频道和将在俄亥俄州西南部播出的“ThinkTV”节目教育公众关于微纳米技术领域对医学的重要性。外泌体是直径约30-120 nm的细胞外囊泡，从许多细胞类型释放到细胞外空间。它们由含有各种受体和四跨膜蛋白的脂质双层膜组成。它们还将核酸、蛋白质和脂质包封在其管腔中。外泌体是有前途的生物标志物，原因有几个：1）它们在所有体液中高度丰富，因此容易获得; 2）它们的组成反映了它们的细胞起源，因此可以作为病理学的指标; 3）它们是稳定的。此外，已经显示，从不同细胞来源分泌的外泌体，特别是致病性外泌体，经历组成变化，并且可能具有额外的膜受体和/或升高或抑制的核酸水平，这可能与它们的总电荷和偶极子有关。然而，由于缺乏可行的技术来及时可靠地分离和严格表征其独特性质，因此将外来体用作生物标志物受到阻碍。 虽然外来体的一些生物物理性质，如大小，密度和形态已经被表征，但与其独特的组成电荷相关的介电性质尚未被研究。所提出的无标记芯片实验室装置将利用硼硅酸盐微量移液器阵列上的可控动电力，在移液器尖端快速捕获外来体，并通过测量其阻抗，基于其独特的介电特性来表征囊泡。当在宽频谱范围（500 KHz至50 MHz）下施加交流电流（AC）时，将通过嵌入移液器吸头附近的微电极阵列进行阻抗测量。在宽频率范围内的AC场将消除与外泌体结构相关的束缚和未束缚电荷。阻抗测量的差异将与外泌体独特的介电性质有关，包括它们的膜电容和胞质电导。此外，将测量从不同细胞来源和大小分布分泌的外泌体的阻抗，并研究其介电特性的独特性。这种快速和无标记的电动装置可以进一步发展为病原性外泌体的初始非侵入性检测的诊断工具，同时保持其成分完整，以进行进一步的下游分析。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

项目成果

A Label-Free Electrical Impedance Spectroscopy for Detection of Clusters of Extracellular Vesicles Based on Their Unique Dielectric Properties.

无标记的电阻抗光谱，用于根据其独特的介电特性检测细胞外囊泡的簇。

• DOI: 10.3390/bios12020104
• 发表时间: 2022-02-09
• 期刊: Biosensors
• 作者: Zhang Y;Murakami K;Borra VJ;Ozen MO;Demirci U;Nakamura T;Esfandiari L
• 通讯作者: Esfandiari L